The generation of transgenic zebrafish to study podocyte biology

The Global Burden of Disease Study has shown that kidney disease is one of the fastest rising causes of mortality worldwide and claims 1.1 million lives per year. A range of factors can cause kidney damage. The kidneys filter approximately 180 litres of blood per day, and a decline in kidney function is quickly followed by a severe decline in health and eventually death. A late-stage indicator of kidney disease is proteinuria, and a common feature connecting many kidney diseases is damage to podocyte cells. Podocyte cells are a vital cell type involved in blood filtration and the maintenance of kidney function. Podocytes are highly specialised cells that envelope the glomerular capillaries and form part of a selective barrier, ensuring the retention of larger proteins within the lumen of the capillaries they encapsulate. Increasing the understanding of podocyte cell physiology will allow us to gain novel insight, improve our knowledge of cellular interactions within podocytes, and hopefully lead to novel therapeutic techniques. Elucidating the physiology of the kidney will enable the development of improved therapies and treatments for managing kidney diseases. In this study, novel transgenic zebrafish podocyte reporter lines have been created as informative tools in analysing podocyte cell function and changes within podocytes. The rapid external development of larvae and the optical clarity of zebrafish made it an ideal model for this project, where observation and analysis of podocyte cells in vivo and the ability to image the transparent larvae was an essential aspect of this work. In this study, using a range of advanced imaging techniques and the analysis of the reporter lines, it has been possible to capture changes to podocyte cells. The overarching aim of this work was to gain greater insight into podocyte physiology. Using gateway cloning, informative, podocyte-specific, transgenic zebrafish lines were generated and used as tools to study podocyte cells. These lines used podocyte-specific fluorescent proteins to identify and locate podocytes within larvae and adults, allowing these cells to be targeted, isolated, and monitored in experimental use. Using SPIM (Single/ Selective Plane Illumination Microscopy), it was possible to induce injury within podocytes of larval fish and visualise changes in calcium ion dynamics in real-time. Through the microinjection of the known nephrotoxic agent Puromycin aminonucleoside, it was possible to induce oedema in larval fish and assess the morphological changes to podocyte cells through analysis via transmission electron microscopy. Using the novel GCaMP6s LifeActTagRFP-T zebrafish line the structure of the actin cytoskeleton was assessed in closer detail. Bioinformatics approaches were used to develop a pathway diagram elucidating the interactions within podocyte cells through manual curation of literature, the use of current datasets, and incorporating novel scRNA seq data. This detailed diagram presents an interactive network that allows the visualisation of podocyte cell interactions in a way that is more accessible and permits visualisation of the data in ways that would not be possible with graphs and images alone. It allows for the identification of discrepancies in metabolic pathways and intracellular communications to be displayed; it also contributes to what is currently known about podocyte physiology. Finally, using GCaMP6s and LifeActTagRFP-T tagged podocyte cells disassociated from adult fish, it was possible to generate a single-cell RNA sequencing (scRNA-seq) library, detailing active transcripts present within healthy zebrafish podocytes and the identification of distinct cell populations and podocyte cell markers, adding to and allowing comparisons with libraries generated from zebrafish podocytes in alternative states, such as having undergone injury, or in alternative species. The combination of these techniques and outcomes of this work has allowed the presentation of a project which has contributed innovative transgenic zebrafish reporter lines, the novel display of an increase of calcium ion concentration in injured podocyte cells, the production of a detailed, novel scRNA seq library and a pathway diagram utilising bioinformatics techniques, that is interactive and informative. These contributions can add to the generation of new therapies for treating cardiovascular diseases and have provided additional transgenic zebrafish lines to be used as a model to study kidney disease development, injury, and repair. The Global Burden of Disease Study has shown that kidney disease is one of the fastest rising causes of mortality worldwide and claims 1.1 million lives per year. A range of factors can cause kidney damage. The kidneys filter approximately 180 litres of blood per day, and a decline in kidney function is quickly followed by a severe decline in health and eventually death. A late-stage indicator of kidney disease is proteinuria, and a common feature connecting many kidney diseases is damage to podocyte cells. Podocyte cells are a vital cell type involved in blood filtration and the maintenance of kidney function. Podocytes are highly specialised cells that envelope the glomerular capillaries and form part of a selective barrier, ensuring the retention of larger proteins within the lumen of the capillaries they encapsulate. Increasing the understanding of podocyte cell physiology will allow us to gain novel insight, improve our knowledge of cellular interactions within podocytes, and hopefully lead to novel therapeutic techniques. Elucidating the physiology of the kidney will enable the development of improved therapies and treatments for managing kidney diseases. In this study, novel transgenic zebrafish podocyte reporter lines have been created as informative tools in analysing podocyte cell function and changes within podocytes. The rapid external development of larvae and the optical clarity of zebrafish made it an ideal model for this project, where observation and analysis of podocyte cells in vivo and the ability to image the transparent larvae was an essential aspect of this work. In this study, using a range of advanced imaging techniques and the analysis of the reporter lines, it has been possible to capture changes to podocyte cells. The overarching aim of this work was to gain greater insight into podocyte physiology. Using gateway cloning, informative, podocyte-specific, transgenic zebrafish lines were generated and used as tools to study podocyte cells. These lines used podocyte-specific fluorescent proteins to identify and locate podocytes within larvae and adults, allowing these cells to be targeted, isolated, and monitored in experimental use. Using SPIM (Single/ Selective Plane Illumination Microscopy), it was possible to induce injury within podocytes of larval fish and visualise changes in calcium ion dynamics in real-time. Through the microinjection of the known nephrotoxic agent Puromycin aminonucleoside, it was possible to induce oedema in larval fish and assess the morphological changes to podocyte cells through analysis via transmission electron microscopy. Using the novel GCaMP6s LifeActTagRFP-T zebrafish line the structure of the actin cytoskeleton was assessed in closer detail. Bioinformatics approaches were used to develop a pathway diagram elucidating the interactions within podocyte cells through manual curation of literature, the use of current datasets, and incorporating novel scRNA seq data. This detailed diagram presents an interactive network that allows the visualisation of podocyte cell interactions in a way that is more accessible and permits visualisation of the data in ways that would not be possible with graphs and images alone. It allows for the identification of discrepancies in metabolic pathways and intracellular communications to be displayed; it also contributes to what is currently known about podocyte physiology. Finally, using GCaMP6s and LifeActTagRFP-T tagged podocyte cells disassociated from adult fish, it was possible to generate a single-cell RNA sequencing (scRNA-seq) library, detailing active transcripts present within healthy zebrafish podocytes and the identification of distinct cell populations and podocyte cell markers, adding to and allowing comparisons with libraries generated from zebrafish podocytes in alternative states, such as having undergone injury, or in alternative species. The combination of these techniques and outcomes of this work has allowed the presentation of a project which has contributed innovative transgenic zebrafish reporter lines, the novel display of an increase of calcium ion concentration in injured podocyte cells, the production of a detailed, novel scRNA seq library and a pathway diagram utilising bioinformatics techniques, that is interactive and informative. These contributions can add to the generation of new therapies for treating cardiovascular diseases and have provided additional transgenic zebrafish lines to be used as a model to study kidney disease development, injury, and repair.